Quasispecies Level Research Articles

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.

Obesity: A neurobiological approach

A huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016–2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure.We performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines.HAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation.Our data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group.

Evidence for positive selection of hepatitis A virus antigenic variants in vaccinated men-having-sex-with men patients: Implications for immunization policies.

BackgroundA huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016–2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure.MethodsWe performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines.FindingsHAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation.InterpretationOur data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group.

Towards Personalized Medicine: An Improved De Novo Assembly Procedure for Early Detection of Drug Resistant HIV Minor Quasispecies in Patient Samples.

The third-generation sequencing technology, PacBio, has shown an ability to sequence the HIV virus amplicons in their full length. The long read of PaBio offers a distinct advantage to comprehensively understand the virus evolution complexity at quasispecies level (i.e. maintaining linkage information of variants) comparing to the short reads from Illumina shotgun sequencing. However, due to the highnoise nature of the PacBio reads, it is still a challenge to build accurate contigs at high sensitivity. Most of previously developed NGS assembly tools work with the assumption that the input reads are fairly accurate, which is largely true for the data derived from Sanger or Illumina technologies. When applying these tools on PacBio high-noise reads, they are largely driven by noise rather than true signal eventually leading to poor results in most cases. In this study, we propose the de novo assembly procedure, which comprises a positivefocused strategy, and linkage-frequency noise reduction so that it is more suitable for PacBio high-noise reads. We further tested the unique de novo assembly procedure on HIV PacBio benchmark data and clinical samples, which accurately assembled dominant and minor populations of HIV quasispecies as expected. The improved de novo assembly procedure shows potential ability to promote PacBio technology in the field of HIV drug-resistance clinical detection, as well as in broad HIV phylogenetic studies.

Quasispecies composition and diversity do not reveal any predictors for chronic classical swine fever virus infection.

Classical swine fever (CSF) can run acute, chronic, and prenatal courses in both domestic pigs and wild boar. Although chronic infections are rare events, their epidemiological impact is very high due to the long-term shedding of virus. So far, little is known about the factors that influence disease course and outcome from either the host or virus's perspective. To elucidate the viral determinants, we analyzed the role of the viral populations for the development of chronic CSF virus (CSFV) infections. Three different animal trials that had led to both chronic and acute infections were chosen for a detailed analysis by deep sequencing. The three inocula represented sub-genogroups 2.1 and 2.3, and two viruses were wild-type CSFV, one derived from an infectious cDNA clone. These viruses and samples derived from acutely and chronically infected animals were subjected to next-generation sequencing. Subsequently, the derived full-length genomes were compared at both the consensus and the quasispecies level. At consensus level, no differences were observed between the parental viruses and the viruses obtained from chronically infected animals. Despite a considerable level of variability at the quasispecies level, no indications were found for any predictive pattern with regard to the chronicity of the CSFV infections. While there might be no direct marker for chronicity, moderate virulence of some CSFV strains in itself seems to be a crucial prerequisite for the establishment of long-term infections which does not need further genetic adaption. Thus, general host and virus factors need further investigation.

Hepatitis B virus full-length genomic mutations and quasispecies in hepatocellular carcinoma.

Hepatitis B virus (HBV) full-length genomic mutations and quasispecies characteristics in hepatocellular carcinoma (HCC) were investigated. Hepatitis B virus DNA was extracted from the tumor and non-tumor tissues of 16 HCC patients. Overlapping DNA fragments covering the entire HBV genome were amplified and sequenced. To study HBV sequence at the quasispecies level, the preS region was amplified and clonally sequenced. HBV mutation profiles, quasispecies complexity and diversity, and phylogenetic characteristics were assessed. Fourteen patients had full-length HBV amplification. Hot-spot mutations at HBx aa130-131 and pre-S deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C regions were more frequently detected in the tumor than in the non-tumor tissues (P = 0.031). Compared with the non-tumor tissues, the tumor tissues had a lower quasispecies complexity (P = 0.014 and 0.043, at the nucleotide and amino acid levels, respectively) and diversity (P = 0.048 and 0.022, at the nucleotide and amino acid levels, respectively). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by the correlation coefficient testing on both the number and length of branches of viral populations (all P < 0.02). Hepatitis B virus mutation patterns in HCC tumor tissues and non-tumor tissues were different. HBV quasispecies within the preS region were compartmentalized, and tumor tissues had a lower genome complexity and diversity. Our study suggests HBV evolution is conditioned by the differential host cellular environment in HCC tumors.

Tracing hepatitis C virus transmission by using high-throughput sequencing technique

An approach for analysis of hepatitis C virus (HCV) quasispecies using Hiseq high-throughput sequencing (hereinafter referred to as Hiseq sequencing) technique was developed and then applied to investigate a possible case of HCV needle sharing transmission. One case of HCV antibody seroconversion (P1) was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users (IDUs), P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period (after March 24, 2014) that P1 may be infected with HCV were investigated, and another 28 HCV antibody positive IDUs were selected as controls (C1 to C28). These controls came from the same methadone clinic or lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies (263 bp) in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated. The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 (average 869 608) cleaned sequences representing 3 to 172 (average 48) unique HCV quasispecies were obtained. The medians (P50) of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 (P25, P75) of genetic diversities between P1 and the other 8 specimens were 19.0% (18.4%, 19.8%), 10.4% (2.8%, 18.3%), 19.6% (17.8%, 21.4%),24.9% (23.8%, 26.1%), 19.8% (18.7%, 20.7%), 20.1% (18.9%, 21.2%), 20.6% (20.0%, 21.1%), 23.6% (22.4%, 24.8%). There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specimens (H=9.40, P=0.100). The genetic diversities between few HCV quasispecies from P1 and few ones from C7 were 0. Phylogenetic tree analysis indicated that there was no HCV transmission relationship between P1 and P2, but there was HCV transmission relationship between P1 and C7. With the feature of high-throughput, easier operation and lower cost, Hiseq sequencing technique has high practical value in tracing HCV transmission at the quasispecies level.

Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients’ HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

Evolution of HIV-1 tropism at quasispecies level after 5 years of combination antiretroviral therapy in patients always suppressed or experiencing episodes of virological failure.

Tropism evolution of HIV-1 quasispecies was analysed by ultra-deep pyrosequencing (UDPS) in patients on first-line combination antiretroviral therapy (cART) always suppressed or experiencing virological failure episodes. Among ICONA patients, two groups of 20 patients on cART for ≥5 years, matched for baseline viraemia and therapy duration, were analysed [Group I, patients always suppressed; and Group II, patients experiencing episode(s) of virological failure]. Viral tropism was assessed by V3 UDPS on plasma RNA before therapy (T0) and on peripheral blood mononuclear cell proviral DNA before-after therapy (T0-T1), using geno2pheno false positive rate (FPR) (threshold for X4: 5.75). For each sample, quasispecies tropism was assigned according to X4 variant frequency: R5, <0.3% X4; minority X4, 0.3%-19.9% X4; and X4, ≥20% X4. An R5-X4 switch was defined as a change from R5/minority X4 in plasma/proviral genomes at T0 to X4 in provirus at T1. At baseline, mean FPR and %X4 of viral RNA were positively correlated with those of proviral DNA. After therapy, proviral DNA load significantly decreased in Group I; mean FPR of proviral quasispecies significantly decreased and %X4 increased in Group II. An R5-X4 switch was observed in five patients (two in Group I and three in Group II), all harbouring minority X4 variants at T0. UDPS analysis reveals that the tropism switch is not an 'on-off' phenomenon, but may result from a profound re-shaping of viral quasispecies, even under suppressive cART. However, episodes of virological failure seem to prevent reduction of proviral DNA and to accelerate viral evolution, as suggested by decreased FPR and increased %X4 at T1 in Group II patients.

P141 Viral genome sequencing and small RNA detection by next generation sequencing

Introduction Next generation sequencing technologies are increasingly used in many domains. Their application to virology opens the door to various investigations such as virus sequencing and genotyping; analysis at quasispecies level; identification of causative agents of undiagnosed diseases; and finally study of host responses induced by viral infections. In this study, we used influenza- and rhinoviruses as models to validate some of these applications. Methods First we used influenza A virus infected cells to compare complete genome coverage after purification and ultra-deep sequencing of small RNAs (20–30 nt) or total fragmented RNAs (150–250 nt). Second, we sequenced small RNA population in rhinovirus (HRB-B14 and HRV-A39) infected HeLa cells and analyzed human micro RNA (miRNA) changes and the distribution of sequence reads on the viral genome. Results As expected the number of viral reads and genome coverage were much higher by sequencing total RNA, despite a lower ratio of viral versus host sequences. In contrast, small RNA purification from rhinovirus infected cells allowed us to point out to miRNA expression changes upon viral infection as well as over-expressed small viral RNAs (svRNAs). Conclusion svRNAs match the rhinovirus 5′UTR cloverleaf region, an important structural element implicated in the regulation of replication. The generation and the functional role of these svRNAs is currently under investigation.

Co-receptor usage prediction at quasispecies level using ultra-deep pyrosequencing on both circulating and proviral hiv in patients candidates to CCR5 antagonist treatment

Methods Seventeen patients were enrolled. Trofile phenotypic test was performed by standard in 9 and by enhanced procedures in 8 patients. UDPS was carried out on both plasma virus RNA and on PBMC proviral DNA, amplifying env V3 loop region. Genotypic prediction of coreceptor usage was obtained by Position Specific Score Matrix analysis. Comparison between phenotypic and genotypic assays was performed considering a threshold of 5% and 0.3% for X4 detection (standard and enhanced Trofile sensitivity).

Hepatitis B precore/core promoter mutations in isolates from HBV-monoinfected and HBV–HIV coinfected patients: A 3-yr prospective study

Background The course of chronic HBV infection is modified by HIV-coexistence. Objective To analyze the role of HBV genomic heterogeneity in basal core promoter (BCP) and precore (Pc) genomic regions. Study design In a 3-yr prospective study, 39 HBV infected patients (20 monoinfected and 19 HIV-coinfected) were included. Eighty-two HBV isolates were studied at quasispecies level in the BCP/Pc genomic region. Clinical records obtained include data on lamivudine therapy and resistance mutations, HBV and HIV-viral load. Results HBV isolates were predominantly ascribed to genotype (Gt) A2 among HBV-monoinfected and HIV-coinfected patients. BCP mutations in isolates from monoinfected patients were significantly more frequent than in those from coinfected ones, irrespective of the HBe expression pattern ( p < 0.0001). Regardless of the HIV-coexistence, the Pc mutation at G1896A only barely appeared among clone-derived sequences of GtF1 isolates, mainly from HBe(−) HBV-monoinfected patients. Conclusions HBV isolates characterized from HIV-coinfected patients seem to be more prone to exhibit a wild type genomic pattern at BCP regulatory region with respect to those from HBV-monoinfected ones. Besides, mutations at Pc region might be genotype-dependent in their frequency but not on HIV co-presence related.

Separation of near full-length hepatitis C virus quasispecies variants from a complex population

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163–1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.

Evolutionary Dynamics of the Glycan Shield of theHuman Immunodeficiency Virus Envelope during Natural Infection andImplications for Exposure of the 2G12Epitope

Elucidation of the kinetics of exposure of neutralizing epitopes on the envelope of human immunodeficiency virus type 1 (HIV-1) during the course of infection may provide key information about how HIV escapes the immune system or why its envelope is such a poor immunogen to induce broadly efficient neutralizing antibodies. We analyzed the kinetics of exposure of the epitopes corresponding to the broadly neutralizing human monoclonal antibodies immunoglobulin G1b12 (IgG1b12), 2G12, and 2F5 at the quasispecies level during infection. We studied the antigenicity and sequences of 94 full-length envelope clones present during primary infection and at least 4 years later in four HIV-1 clade B-infected patients. No or only minor exposure differences were observed for the 2F5 and IgG1b12 epitopes between the early and late clones. Conversely, the envelope glycoproteins of the HIV-1 quasispecies present during primary infection did not expose the 2G12 neutralizing epitope, unlike those present after several years in three of the four patients. Sequence analysis revealed major differences at potential N-linked glycosylation sites between early and late clones, particularly at positions known to be important for 2G12 binding. Our study, in natural mutants, confirms that the glycosylation sites N295, N332, and N392 are essential for 2G12 binding. This study demonstrates the relationship between the evolving "glycan shield " of HIV and the kinetics of exposure of the 2G12 epitope during the course of natural infection.

Dynamics of rabies virus quasispecies during serial passages in heterologous hosts.

To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21.9 x 10(-4) mutations per bp), noncoding sequence nucleoprotein-phosphoprotein region (7.2-7.9 x 10(-4) mutations per bp) and nucleoprotein gene region (2.9-3.7 x 10(-4) mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. The shape of the quasispecies structure was dramatically modified by passages in mice, in which the mutation frequencies increased by 12-31 x 10(-4) mutations per bp, depending on the region considered. Non-synonymous mutations were preponderant particularly in the glycoprotein gene, stressing the importance of positive selection in the maintenance and fixation of substitutions. Two mechanisms of genomic evolution of the rabies virus quasispecies, while adapting to environmental changes, have been identified: a limited accumulation of mutations with no replacement of the original master sequence and a less frequent but rapid selective overgrowth of favoured variants.